The advent of CRISPR-based genome modification has lowered the investment cost required for the creation of highly-customized model organisms. Here we describe the development of a Cas9-based genetic toolkit for the rat, which streamlines the targeted insertion of transgenes into the Rosa26 locus. We have constructed a set of plasmids which express the Cas9(D10A) nickase (Cas9n) and a pair of rat Rosa26-targeting guide RNAs (gRNAs) along with a selectable marker. When transfected into rat adrenal gland pheochromocytoma (PC-12) cells, these plasmids induce the formation of double-strand breaks and thereby lead to the accumulation of small insertion/deletions (InDels) between the gRNA binding sites. These plasmids also increase the efficiency of homology-directed insertion of a transgene (donor template) when delivered by cotransfection. The verified Rosa26-targeting gRNAs were transcribed and purified in vitro, and microinjected alongside Cas9n mRNA into Long-Evans rat embryos. InDel frequency at the Rosa26 locus in the resulting pups was assessed using a T7 endonuclease assay. For three rounds of injections, the frequency of InDels detected was (2 out of 16), (7 out of 12), and (10 out of 15). The ability to enhance homology-directed insertions in vivo was also validated by coinjecting these gRNAs with single-stranded oligonucleotides (ssODN) containing a novel loxP site flanked on each side by 72 basepairs of Rosa26 sequence. A single round of injections produced 15 pups, 4 of which carried the loxP allele. Next, a single round of injections employing a plasmid-based donor template with 1 kilobase homologous arms produced 7 pups, 1 of which carried the transgene. The use of this rat Rosa26 CRISPR toolkit will simplify the creation of “defined” transgenic rat models and should reduce the inconsistencies attributed to copy number- and position-effects when dealing with randomly integrated transgenes.